Pharmacokinetics and tissue residues of albendazole sulphoxide and its metabolites in donkey after intramuscular injection

Abstract Background Various anti‐parasitic drugs are used to control donkey parasitic diseases. The abuse of donkey drugs leads to the disposition of residues in the edible parts of treated donkeys. Objectives The aim of this study was to (1) analyse the pharmacokinetics of ABZSO to serve as reference for the dosage regimen in donkey; and (2) calculate the withdrawal times of the ABZSO in the tissue of the donkey. Methods The concentrations of ABZSO and its metabolites in plasma and tissues were determined using high‐performance liquid chromatography with an ultraviolet detector. Pharmacokinetic analysis was performed by the programme 3p97. Results The plasma concentrations of ABZSO and ABZSO2 concentration–time data in donkey conformed to the absorption one‐compartment open model. The t1/2ke of ABZSO was 0.67 h, whereas the t 1/2 k e was 12.93 h; the C max and the T p were calculated as 0.58 μg mL−1 and 3.01 h. The V d/F of ABZSO was estimated to be 10.92 L kg−1; the area under the curve (AUC) was 12.81 μg mL−1 h. The C max and AUC values of ABZSO were higher than those of ABZSO2; however, t 1/2 K e and V d/F were lower. Other pharmacokinetics parameters were similar between the two metabolites. Conclusions The results revealed that ABZSO2 was the main metabolite of ABZSO in donkey plasma. The concentrations of ABZSO and its chief metabolite (ABZSO2) were detected in liver, kidney, skin and muscle; however, ABZ‐SO2NH2 was only detected in liver and kidney. The results also revealed that the depletion of ABZSO and its metabolite in donkey was longer, especially in skin.

broad-spectrum anti-parasitic agents used against the most important animal gastrointestinal helminth species (Son et al., 2020).The main metabolites of albenzdazole (ABZ) were albendazole sulphoxide (ABZSO), albendazole sulfone (ABZSO 2 ) and albendazole-2aminosulfone (ABZ-SO 2 NH 2 ).ABZSO is the main anthelmintically active metabolite recovered in blood (Theurillat et al., 2021;Zhang et al., 2020) and tissues (Busatto et al., 2018;Pereira Cordeiro et al., 2021), after treatment with ABZ in cattle and sheep.Pharmacokinetics of ABZSO has been extensively studied in other species, including humans (Ceballos et al., 2018;Myers et al., 2021;Paredes et al., 2018).Donkey is one of the most important herbivores in the world, and it is easy to suffer from parasitic disease.Chemotherapy is widely used to control Mammalian parasitic infection.ABZSO is an available anthelmintic compound in ruminant species as an oral preparation and, more recently, as an injectable formulation in cattle (Feng et al., 2017).Now it is also gradually used to control donkey parasitic diseases.Lixin have studied the mice infected with Echinococcus granulosus treatment for ABZSO, and the results showed that ABZ is secondary to ABZSO in the anthelmintic efficacy.The reason is that the solubility of HCL-ABZSO is greater than ABZ in intestinal and then has good absorption (Pavan et al., 2007).ABZSO is allowed to be used for the prevention and treatment of parasitic diseases in donkeys in China.So HCL-ABZSO would have a good prospect of application in donkey.
As the use of benzimidazol anthelmintics in animal production industries has been increasing over the last two decades, what is more, the problems which the abuse of drugs leads to residues have become serious.However, toxicological studies in both farm and laboratory animals have shown ABZ and ABZSO could be teratogenic (Belew et al., 2021;Portela et al., 2020).'Maximum Residue Limits for Veterinary Drugs in Animal Food in China' stipulates the total amount of ABZSO, ABZSO 2 and ABZ-SO 2 NH 2 as residual markers of ABZ in animal food (muscle, fat: 100 µg kg −1 ; liver, kidney: 5000 µg kg −1 ).In order to guide the use of HCL-ABZSO for the treatment of donkey disease in the farming of donkey, information was needed on the pharmacokinetics in donkey.Nevertheless, no data is available on the pharmacokinetics of HCL-ABZSO in donkey.Studies on the pharmacokinetics of HCL-ABZSO were performed in cattle, horse and fish (Dar et al., 2017;Li et al., 2012;Myers et al., 2021).As the parameters of pharmacokinetics and residues are variable in different species, it is unreasonable that HCL-ABZSO data is used directly in donkey.
This study was undertaken to research pharmacokinetics and tissue residues of HCL-ABZSO in donkey.The objectives of this study are (1) to analyse the pharmacokinetics of ABZSO, to serve as reference for the dosage regimen in donkey; (2) to calculate the withdrawal times (WDTs) of the ABZSO in the tissue of the donkey.

Chemicals
All agents used for the analysis were of the high-performance liquid
Five donkeys were used for pharmacokinetics experiment, and 55 animals were used for residues experiment.The donkeys were fed antibiotic-free feed (silage corn) twice a day, and water was available.
All animals remained in good health during the experiments.

Drug administration and sampling
During the study of pharmacokinetics by IM injection, donkeys were given a single dose of 7.5 mg kg −1 BW ABZSO of a solution with dimethyl sulphoxide.In the residues study, donkeys were given a multidose of 7.5 mg kg −1 BW for 3 consecutive days by IM injection.
After IM injection administration in the study of pharmacokinetics, five donkeys were sampled at each time-point (Table 1), using evacuated syringes containing heparin to prevent clotting.The plasma was stored at −20 • C.
For the purpose of the residue study of ABZSO, tissues, such as liver, muscle, kidney and skin, were sampled at 4,8,12,24,48,72,96,120,144,168 and 240 h after the last administration.The tissues of donkey were collected at each time-point.Control samples (n = 5) were collected alternately from the control groups.All samples were stored at −20 • C.

Sample analysis
The HPLC system was Shimadzu LC-2030 series equipment, with a variable wavelength detector.ABZSO and its metabolite detection were performed at 290 nm.An automatic injection of 20 µL was performed on SB-C 18 stainless steel column (250 × 4.6 mm 2 , 5 µm).
Thermostatted column compartment was used to maintain the column temperature at 32 • C. The mobile phase consisted of acetonitrile and 0.02 mol L −1 ammonium acetate solution (pH = 4.8) (24:76, v/v), and the flow rate was 1.0 mL min −1 .
TA B L E 1 ABZSO and its metabolites concentrations in plasma following IM injection (n = 5).After samples were thawed at room temperature, the extraction of ABZSO and its metabolites in plasma and tissues was carried out.

Mean drug concentration (µg mL
A sample of 4 mL plasma was placed in a 15 mL plastic centrifuge tube.A 0.4 mol L −1 sodium hydroxide solution of 1000 µL and 10 mL of ethylacetate used as extractant were added; the mixture was vortexed for 2 min and then centrifuged for 10 min at 3000 rpm.The supernatant was removed to a fresh tube and dried at 70 • C under nitrogen.Extraction of ABZSO and its metabolites in muscle, skin, liver and kidney samples was carried out according to the method of plasma analysis.The homogenate (4.0 g) was transferred to a 15 mL plastic centrifuge tube.The samples were extracted three times using ethylacetate (10 mL) in order to achieve high recovery.The extracts were combined and dried at 70 • C under nitrogen.The residue was reconstituted in 200 µL of the mobile phase and then degreased by N-hexane before analysis.

Method validation
The method was validated for plasma, muscle, skin, liver and kidney tissues.The standard calibration curves were prepared within the concentrations of 0.05, 0.1, 0.2, 0.5, 2 and 5 µg mL −1 in plasma and 0.05, 0.1, 0.5, 1, 5 and 10 µg g −1 in all tissues.Linearity, recovery, intra-and inter-assay precisions and accuracies were determined by the standard curve.Recovery was calculated by comparing the peak area of the drugs from processed samples with that from the drug standard in the mobile phase.Accuracy was determined by comparing the measured concentration to its true value.The variability in the peak area ratios at each concentration was determined as an indicator of the precision.The limit of detection (LOD) and the limit of quantitation of the drugs were defined as the drug concen-trations resulting in a peak height 3 and 10 times the signal noise, respectively.

Pharmacokinetic and residue analysis
Pharmacokinetic analysis was performed by the programme 3p97 (version 1.0, edited by the Chinese Pharmacological Society).Following a single dose of an IM injection administration, the pharmacokinetic parameters of ABZSO and ABZSO 2 were both calculated according to the one-compartment open models with first-order rate processes, using the following equation: where C is the plasma concentration at any time (t); M is zero time intercept of the elimination phase; k e is the elimination rate constant; K a is the absorption rate constant.
The area under the curve (AUC) was determined according to the following equations: AUC (µg mL The volume of distribution was calculated from the equation: Total body clearance (CL b ) was calculated using the following equations: The elimination half-life (t1∕) was determined by the following equation: In the residues study, the elimination characteristic of the drug from each tissue was estimated according to first-order rate processes using the equation: C = C 0 e −kt , where C is the tissue concentrations of ABZSO and its metabolites at time t; C 0 is an extrapolate concentration of ABZSO and its metabolites in target tissues after IM injection; k is the elimination rate constant.The data was analysed by the least squares method.The elimination half-life (t1∕) was calculated from the equation: t 1/2ke = 0.693/k.The WDT would be calculated by WT1.4 software according to GB 31650-2019, which formulated the residue marker for ABZ in food animals (ABZ-SO 2 NH 2 ) and the maximum residue limit (muscle: 100 µg kg −1 ; fat: 100 µg kg −1 ; liver: 5000 µg kg −1 ; kidney: 5000 µg kg −1 ).

Method validation
The values of correlation coefficient (r), recovery, accuracy, precision of inter-and intra-days and LOD of the analytical method for ABZSO and its metabolites in all tissues are listed in Table 2.

TA B L E 2
The method validation in plasma, muscle, liver, akin and kidney.

Pharmacokinetics
ABZSO and ABZSO 2 were recovered in plasma between 0.5 and 72 h after ABZSO IM injection administration.ABZ-SO 2 NH 2 was not detected in plasma at any time.Pharmacokinetic parameters of ABZSO and its chief metabolite (ABZSO 2 ) in donkey obtained after IM injection with HCL-ABZSO of 7.5 mg kg −1 are shown in Table 3.
The plasma concentrations of ABZSO and ABZSO 2 concentrationtime data in donkey conformed to the absorption one-compartment open model.The maximum plasma concentration (C max ) of ABZSO with 0.58 µg mL −1 was obtained at 3.01 h, compared to ABZSO 2 (0.43 µg mL −1 at 3.14 h), respectively.The absorption half-lives (t1∕) of ABZSO were calculated to be 0.67 h, whereas the elimination halflives (t1∕) was 12.93 h.The distribution volume (V d /F) of ABZSO was estimated to be 10.92 L kg −1 , whereas that of ABZSO 2 was 15.07 L kg −1 .The total clearance (CL b ) of ABZSO was computed as 0.59 L (h kg) −1 , and that of ABZSO 2 was 0.72 L (h kg) −1 .The area under the concentration-time curve (AUC) of ABZSO was 12.81 63.12 µg mL −1 h, compared to ABZSO 2 (10.38 µg mL −1 ), respectively.
F I G U R 1 Albendazole sulphoxide (ABZSO) and albendazole sulfone (ABZSO 2 ) levels in plasma of donkey after a single IM administration (7.5 mg kg −1 ).
Plasma concentration of ABZSO and ABZSO 2 vs. time curve detected in donkey is given in Figure 1.

Residues
ABZSO and its metabolite concentrations in liver, kidney, skin and muscle are listed in Tables 4-7, respectively.It was obvious that the t1∕ in skin was longer than that in other tissues.ABZSO and ABZSO 2 were detected in all tissues.However, ABZSO 2 -NH 2 was only detected in liver and kidney, which was not detected in muscle and skin.The concentration of ABZSO and its metabolites was found to be higher in liver than that in other tissues.However, it decreased rapidly in liver after the 24 h post-administration.ABZSO and its metabolite concentration-time curves in all tissues are given in Figure 2.
The results reveal that the WDT was 3 days calculated by WT1.4 software according to GB 31650-2019.Compared with the other tissues, the concentrations of ABZSO and its metabolites were higher in liver at each time-point, whereas the depletion of ABZSO and its metabolite in skin was longer.Because skin is the edible product of donkey, which is the main raw material of donkey-hide gelatin.The elimination of ABZSO and its metabolites in skin was the slowest, which behaved as a reservoir in donkey.The WDT of ABZSO in donkey should not be less than 3 days when administered by IM injection with a multi-dose of 7.5 mg kg −1 BW.
TA B L E 4 ABZSO and its metabolites concentration in liver (n = 5).

Time (h)
Mean ABZSO and its metabolites concentration (µg g

DISCUSSION
After IM injection administration, ABZSO was rapidly absorbed in donkey.The K a of ABZSO was 1.04 h −1 , which is more rapidly than ABZ directly.Goudah (2003) studied the pharmacokinetics of ABZ and ABZSO in sheep after oral administration.These results showed that the absorption of two drugs is similar, whereas the elimination rate of ABZSO was faster than the ABZ.The data in this experiment was consistent with Goudah's studies.This may be due to its good solubility and easy absorption; however, other benzimidazole anthelmintic solutes lowly and absorps poorly in the intestine.
The drug was in the form of ABZSO mainly, whereas the form of ABZSO 2 is secondary in the plasma, which may be because only a small amount of ABZSO converted into ABZSO 2 and then metabolized to ABZ-SO 2 NH 2 .(Capece et al., 2000) have studied the pharmacokinetics of ABZ and ABZSO in sheep and goats; Formentini et al. (2001) have reported ABZ and ABZSO kinetic dispositions after treatment with different formulations in calves.The results demonstrated that the concentration of ABZSO in plasma was higher than that of ABZSO 2 in these two kinds of animals, which was in accordance with the conclusion of donkey.There was little difference between ABZSO and ABZSO 2 about the peak time (T p ) in donkey, which was consistent with Tp in scalper.
The V d /F of ABZSO was 10.92 L kg −1 , and which of its main metabolite (ABZSO 2 ) was 15.07 L kg −1 .It was assessed that ABZSO and ABZSO 2 were well distributed throughout the body.Because the parasite was generally located in donkey skin, the distribution characteristic in skin could be considered an advantage of ABZSO.
TA B L E 6 ABZSO and its metabolites concentration in skin (n = 5).

Time (h)
Mean ABZSO and its metabolites concentration (µg g There was no significant deviation between ABZSO and ABZSO 2 about the t1∕ in donkey.The t1∕ of ABZSO was a little shorter (12.93 h) than that of ABZSO 2 (14.46).The speed of absorption was rapid, whereas the speed of elimination was slow.The CL b of ABZSO and ABZSO 2 also had no significant deviation, which was 0.59 and 0.72 L (h kg) −1 , respectively.
ABZSO and its metabolite residues were detected in the tissue of the donkey, but GB 31650-2019 formulated the residue marker for ABZ in food animals as ABZ-SO 2 NH 2 .It was indicated that the t1∕ of ABZSO and its metabolites in skin was slower than that in other tissues (p < 0.01).This may be related to the accumulation of drugs in the skin (Benigni et al., 2018).The results indicated that ABZSO 2 was the main metabolite of ABZSO; ABZSO and its main metabolite (ABZSO 2 ) were detected in liver, kidney, muscle and skin, whereas ABZ-SO 2 NH 2 was only detected in liver and kidney, this is in accordance with other mammals (Mortezaei et al., 2019;Pérez-Brígido et al., 2020).The drug in all tissues was eliminated slowly, which could still be detected at 240 h time-point after IM injection.The concentration of ABZSO and its metabolites was highest in skin at this time-point, compared to other tissues.The elimination of ABZSO and its metabolites in donkey skin was slowest, which behaved as a reservoir.Skin is the edible product of donkey, which is the raw material of donkey-hide gelatin, although the residue marker for ABZ in food animals is ABZ-SO 2 NH 2 , which was not detected in skin.
F I G U R E 2 Albendazole sulphoxide (ABZSO) and its metabolites in all tissues of donkey after the last dosing.
Based on the results of the present study, it can be concluded that the elimination of ABZSO and its metabolites in donkey skin was slower than in other tissues, which was in accordance with the research of ABZSO in other mammals.To guarantee that tissue samples are safe for consumption, it is suggested that the WDT of ABZSO in donkey should not be less than 3 days.
ABZSO and its metabolites concentration in kidney in kidney (n = 5).